that silencing a small fraction of the MEC-L2
network is sufficient to interfere with maturation
of the entorhinal-hippocampal circuit only if the
silenced cohort predominantly consists of stellate
cells (Fig. 5). At first glance, this observation is at
odds with the observation in one of the earlier
experiments that maturation requires silencing
of more than 20% of the MEC-L2 cell population
(fig. S6A). One major difference between the two
sets of experiments is that cells infected in utero
were not randomly drawn from the network but
were born on the same day. If isochronic cells act
synergistically during development, then silencing a small but isochronic fraction of the network
might be sufficient to efficiently affect network
maturation. To test this idea, we silenced the isochronic cohort of MEC-L2 excitatory cells born
on E12 and compared the result with the silencing of a comparable fraction of neurons whose
labeling was independent of neurogenesis (fig.
S14A). The E12 isochronic cohort was primed for
silencing in utero and then targeted with the
Cre-dependent hM4D(Gi) receptor at P1 before
delivery of CNO during postnatal development
(fig. S14A, “Isochronic cells”). To label a comparable fraction of neurons randomly, we injected a
mix of AAVs targeting hM4D(Gi) to excitatory
cells postnatally at P1 to avoid any bias to birth
date (fig. S14A, “Random cells”). The viruses were
injected in proportions that allowed labeling of
a sparse cohort of neurons, targeting a fraction of
the L2 network (“Random cells – entire network”)
or a fraction of the stellate cells in MEC-L2 (“
Random cells – stellate cells”), which was comparable
to the isochronic cohort.

We have shown that the entorhinal-hippocampalnetwork matures in a stereotyped and directionalsequence that, with the exception of the DG, re-capitulates the intrinsic excitatory connectivity ofthe transverse hippocampal circuit. At every levelof the circuit, maturation and synaptogenesisrely on an excitatory activity-dependent instruc-tive signal that originates in stellate cells of theMEC and spreads directionally throughout thecircuit over the course of the first month of post-natal life. Stellate cells, being the first to mature,initiate maturation of the network by providingexcitatory drive to their synaptic targets. These,in turn, subsequently exert a driving effect onareas further downstream, resulting in successive,stagewise maturation of the transverse entorhinal-hippocampal circuit. Maturation of stellate cellsthemselves is independent of local and incomingexcitatory activity but correlates with birth date,pointing to cell-autonomous molecular, genetic,or epigenetic pathways, set up before birth, aspotential sources of stellate cell–initiated matu-ration in the entorhinal-hippocampal circuit.

In line with the role that sensory-driven activity
exerts in the primary sensory cortices, stellate cells
might influence computation in the entorhinal-hippocampal network by orchestrating the refinement of connectivity within MEC and hippocampus,
as well as between these structures. By driving
structural maturation of the interneuron network
and of the back projections from the hippocampus, stellate cells might serve as a developmental
teaching layer to ensure strong coupling among
cells that exhibit correlated neural activity in
stimulus space (32). This may be crucial for the
development of attractor network topologies
thought to underlie the formation of grid patterns (10, 32–36).

The progression of neurogenesis and maturation along the dorsoventral MEC axis may account
for the topographic and modular organization of
grid scale in grid cells along this axis. Moreover,
because of the quantal nature of proliferation in
neural progenitor cells (37), neurogenesis may
contribute to discretization of grid cells into modules (38). The production of consecutive generations of “daughter cells” during neurogenesis
may parcel stellate cells into cohorts that each
are composed of neurons born on the same day
(isochronic cells). Such waves of simultaneously
born stellate cells may give rise to parallel networks with unique features, set up in ways that
mirror the formation of parallel microcircuits
of selectively interconnected neurons in the hippocampal trisynaptic loop (26, 39). These subnetworks born at different times might correspond
to the modules of grid cells, which have a similar
anatomical distribution to the cohorts presented
here (38).

The nature of the instructive signal spreadingthrough the network and driving local circuitmaturation remains to be determined. Specificspatial or temporal patterns of depolarizationin the network might be required to elicit post-synaptic responses leading to synaptogenesis andmaturation. One candidate activity pattern is theorchestrated change of calcium concentrationsacross populations of neurons defined as “calciumwaves,” which has been implicated in the estab-lishment of topography in sensory areas (40, 41).In the entorhinal-hippocampal circuit, isochronicneurons may exhibit similarly synchronized activ-ity. Such activity may pattern network maturationstage by stage across the circuit (42). Isochronicneurons might exert a synergistic effect on mat-uration because of their tight temporal couplingduring waves.

Our data show that stellate cells are at the top
of the developmental hierarchy that instructs the
linear sequence of maturation of the entorhinal-hippocampal circuit. Peripheral sensory organs
have long been studied as the source of instructive signals driving the early development of the
forebrain. For example, the olfactory organ instructs the central nervous system to reach its
mature states (43–45), and, in a similar fashion,
thalamocortical axons are involved in cortical
regionalization and the refinement of dendritic
arborization and connectivity in the visual, soma-tosensory, and auditory systems (46, 47). Although
activity from sensory organs provides a clear directionality to the maturation of cortical columns
in sensory areas, we show here that, in the
entorhinal-hippocampal circuit, it is the activity
from stellate cells that drives the maturation of
the entire circuit. Our data identify a network
where such instructive excitatory activity does not
originate from sensory neurons but is provided by
a subpopulation of neurons that functions as an
“autonomous” intrinsic driver in a neurogenesis-dependent manner. The presence of a few of these
intrinsic drivers in the brain during development
might be particularly influential for the coordinated maturation of networks that are positioned
at a great synaptic distance from sensory signals
and extend across multiple areas in the associative
cortices, thereby shaping network topologies supporting higher cognitive function.

Materials and methods

All experiments were performed in accordance
with the Norwegian Animal Welfare Act and the
European Convention for the Protection of Vertebrate Animals used for Experimental and Other
Scientific Purposes, Permit numbers 6021, 6008,
and 7163. C57/Bl6 mice were housed in social
groups of 2-6 individuals per cage under a 12h
light/12h darkness schedule, in a temperature-and humidity-controlled vivarium. Food and water
were provided ad libitum.

Viral injections

For all surgeries, on the day of the injection, anesthesia was induced by placing the subjects in a
plexiglass chamber filled with isoflurane vapor
(5% isoflurane in medical air, flow of 1 liters/
minute). Surgery was performed on a heated surgery table (38°C), air flow was kept at 1 liters/
minute with 1.5–3% isoflurane as determined from
physiological monitoring of vital signs (breathing
and heartbeat). Analgesics were provided immediately before the surgery (Rymadil, Pfizer, 5 mg/kg).
After each procedure, subjects were allowed to recover in a heated chamber (33°C, 30–90 min) until
they regained complete mobility and alertness.

Viral injections at P1

Newborn pups were subjected to viral injection
1 day after birth (P1). Pre-heated ultrasound gel
(39°C, Aquasonic 100, Parker) was generously applied on the pup’s head in order to create a large
medium for the transmission of ultrasound waves.